The overall goal of this project is to define the epigenetic and transcriptional mechanisms underlying cellular reprogramming, a process by which cells switch from a hepatocyte program to a biliary program in vivo. In the liver, correct spatial organization is essential for function, and disruption of normal architecture (by fibrosis) underlies most cases of severe chronic liver disease. The configuration of the biliary tree is of particular importance, and bile duct dysfunction (from obstruction, destruction, or congenital malformation) is a significant cause of liver-associated morbidity and mortality. A clearer picture of how bile ducts are made and remodeled during injury and regeneration will thus have a major impact on our understanding of disease pathogenesis, informing novel strategies for regenerative therapies. In the last grant period, we made several important discoveries related to biliary development, adult cell plasticity, and liver regeneration. Among these was the finding that the Notch pathway - a conserved signaling module known to be important in bile duct development - acts by coordinating biliary differentiation with morphogenesis through a novel tubulogenesis mechanism. Furthermore, we found that newborn and adult hepatocytes retain significant cellular plasticity, exhibiting the ability to undergo in vivo reprogramming (also termed metaplasia or trans-differentiation) to biliary epithelial cells (BECs). Such reprogramming occurs as part of the liver's physiological response to injury and requires Notch signaling. Moreover, Notch itself can drive reprogramming by pushing cells through a stepwise cascade of transcriptional, morphological and functional changes. These discoveries lay the groundwork for the current proposal, with the objective of defining the mechanisms underlying cellular reprogramming in vivo and exploiting these insights for the treatment of cholestatic disease in humans. These objectives will be achieved through the following two interrelated Specific Aims: Specific Aim 1: Determine the molecular mechanisms of hepatocyte-to-biliary reprogramming in vivo. Specific Aim 2: Examine the function and therapeutic potential of biliary reprogramming in vivo. These efforts will provide an unprecedented window into the transcriptional, functional, and epigenetic changes that occur during a change in cell identity in vivo. Importantly, because biliary reprogramming is part of the liver's normal injury response, these insights will have direc physiological relevance. Finally, by using both established mouse models and an innovative human transplantation system in which reprogramming occurs, these studies may provide mechanistic insights that can be used to tip the balance between hepatocytes and BECs, leading to novel treatments for liver disease.